Metabolically engineered E. coli for enhanced production of oxaloacetate-derived biochemicals

Abstract

Metabolic engineering is used to increase the carbon flow toward oxaloacetate to enhance production of bulk biochemicals, such as lysine and succinate, in bacterial fermentations. Carbon flow is redirected by genetically engineering the cells to overexpress the enzyme pyruvate carboxylase.

What is claimed is:  
     
       1. A metabolically engineered  E. coli  cell transformed with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter, wherein said polynucleotide sequence is expressed and produces an enzymatically active pyruvate carboxylase. 
     
     
       2. The metabolically engineered  E. coil  cell of  claim 1  wherein the pyruvate carboxylase is a  Rhizobium etli  pyruvate carboxylase. 
     
     
       3. A metabolically engineered  E. coli  cell transformed with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter; wherein the cell comprises enzymatically active pyruvate carboxylase and wherein the polynucleotide sequence is chromosomally integrated. 
     
     
       4. A method for making a metabolically engineered  E. coli  cell comprising transforming an  E. coli  cell with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter to yield a metabolically engineered  E. coli  cell that comprises enzymatically active pyruvate carboxylase. 
     
     
       5. A method for making succinate comprising: 
       (a) providing a first  E. coli  cell that produces succinate;  
       (b) transforming the first  E. coli  cell with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter to yield a metabolically engineered  E. coli  cell;  
       (c) culturing the metabolically engineered  E. coli  cell to permit expression of the pyruvate carboxylase to cause increased production of succinate relative to a second  E. coli  cell that has not been transformed with the polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter; and  
       (d) isolating the succinate produced by the metabolically engineered  E. coli  cell.  
     
     
       6. The method of  claim 5  wherein step (b) comprises transforming the cell with a nucleic acid fragment comprising an  R. etli  gene encoding pyruvate carboxylase. 
     
     
       7. A method for making succinate comprising: 
       (a) providing a first, metabolically engineered  E. coli  cell that produces succinate, wherein the metabolically engineered  E. coli  cell has been transformed with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter;  
       (b) culturing the metabolically engineered  E. coli  cell under conditions that permit expression of the pyruvate carboxylase to cause increased production of succinate relative to a second  E. coli  cell that has not been transformed with the polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter; and  
       (c) isolating the succinate produced by the metabolically engineered  E. coli  cell.  
     
     
       8. A method for making threonine comprising: 
       (a) providing a firsts metabolically engineered  E. coli  cell that produces threonine, wherein the metabolically engineered  E. coli  cell has been transformed with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter,  
       (b) culturing the metabolically engineered  E. coli  cell under conditions that permit expression of pyruvate carboxylase to cause increased production of threonine relative to a second  E. coli  cell that has not been transformed with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter; and  
       (c) isolating the threonine produced by the metabolically engineered  E. coli  cell.  
     
     
       9. A method for making threonine comprising: 
       (a) providing first  E. coli  cell that produces threonine;  
       (b) transforming the first  E. coli  cell with a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter to yield a metabolically engineered  E. coli  cell;  
       (c) culturing the metabolically engineered  E. coli  cell to permit expression of the pyruvate carboxylase to cause increased production of threonine relative to a second  E. coli  cell that has not been transformed with the polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter; and  
       (d) isolating the threonine produced by the metabolically engineered  E. coli  cell.  
     
     
       10. The method of  claim 9  wherein step (b) comprises transforming the cell with a nucleic acid fragment comprising an  R. etli  gene encoding pyruvate carboxylase.